1. Field of the Invention
The present invention generally relates to the preparation of proteins. In particular, the present invention relates to the preparation and purification of large quantities of at least one colonization factor (CF) and methods of using thereof.
2. Description of the Related Art
Diarrhea caused by enterotoxigenic Escherichia coli (ETEC), commonly referred to as travelers' diarrhea, is a common health problem among travelers visiting less developed or tropical countries. See Peltola, H., et al. (1991) Lancet 338:1285–1289 and Ericsson, C. D. et al. (1993) Clin. Infect. Dis. 16:616–626. Diarrhea caused by ETEC and other ETEC infections are important concerns for military personnel when deployed to less developed countries. See Wolf, M. K., et al. (1993) Clin. Microbiol. 31:851–856 and Bourgeois, A. L., et al. (1993) Am. J. Trop. Med. Hyg. 48:243–248. ETEC may be transmitted by food or water contaminated with animal or human feces. ETEC produces two toxins, a heat-stable toxin (ST) and a heat-labile toxin (LT). ETEC infections may cause profuse watery diarrhea, abdominal cramping, fever, nausea, vomiting, chills, loss of appetite, headache, muscle aches, and bloating.
The current therapy for travelers' diarrhea is to initiate treatment with agents such as bismuth subsalicylate (Pepto-Bismol®), antidiarrheals such as diphenoxylate with atropine (Lomotil®), loperamide HCl (Immodium®), attapulgite (Kaopectate®) and the like, rehydration therapy, and combinations thereof. The majority of the treatments involve the non-specific removal of the offending agents (i.e. toxins) from the intestinal tract. Only in moderate to severe cases of diarrhea where distressing or incapacitating symptoms are reported is antimicrobial therapy recommended. ETEC is frequently resistant to common antibiotics such as trimethoprim-sulfamethoxazole and ampicillin. Fluoroquinolones such as ciprofloxacin have shown some efficacy. Antibiotics are not usually effective at reducing clinical symptoms of the disease and problems associated with antibiotic resistance can occur. Prophylactic use of antibiotics is not recommended. Thus, therapies that specifically remove ETEC from the intestine are needed to provide more effective treatments for ETEC diarrhea.
In order to initiate the infectious process of diarrhea, ETEC must adhere to the host intestinal epithelial cells via the binding between bacterial adhesions, colonization factors (CFs) and host receptors. This binding is commonly referred to as adhesion-receptor interaction. See Beachey, E. H. (1981) J. Infect. Dis. 143:325–345; Satterwhite, T. K., et al. (1978) Lancet 2:181–184; and Warner, L. and Y. S. Kim. (1989) “Intestinal Receptors for Microbial Attachment”, Eds. M. J. G. Farthing, and G. T. Kensch, ENTERIC INFECTION: MECHANISMS, MANIFESTATIONS AND MANAGEMENT, pp. 31–40. Raven Press, New York. ETEC then causes secretory diarrhea by expressing toxins, heat-labile enterotoxin (LT) and heat-stabile enterotoxin (ST). CFs interact with receptors on the host epithelial cells allowing for adherence of the ETEC to the mucosa. See Cassels, F. J. and Wolf, M. W. (1995) J. Indust. Microbiol 15:214–2263. CFs include colonization factor antigens (CFAs), coli surface (CS), and putative colonization factors (PCFs). The CFs that are most commonly expressed by ETEC and therefore targeted as potential vaccine components include CFA/I, CS3 and CS6. See Wolf, M. K. (1997) Clin. Microbiol. Rev. 10(4):569–584; Tacket, C. O., et al. (1994) Vaccine 12(14): 1270–1274; Güereña-Burgueño, F., et al. (2002) Infect. Immun. 70(4):1874–1880; Jertborn, M., et al. (2001) Clin. and Diag. Laboratory Immun. 8(2):424–428; Freedman, D. J., et al. (1998) J. Infect. Dis. 177(3):662–667; and Evans, D. G., et al. (1988) FEMS Micro. Immuno. 117–126. In both natural and in recombinant organisms, the production of CFA/I, CS3, and CS6 is under the control of temperature-regulated promoters.
Unfortunately, prior art methods for large-scale production of such colonization factors (CFs) have been unsatisfactory. In particular, prior art methods resulted in low yields of bacteria and the desired CF under great risks of contamination. For example, U.S. Pat. No. 5,698,416 discloses a process for fermenting and isolating CF based on an example at small scale. However, the levels of purity (70%) obtained by prior art processes were insufficient for approval by the U.S. Food and Drug Administration (FDA) for use in some human vaccine applications. In addition, the yields obtained by prior art processes were not amenable for conducting an FDA approved clinical trial.
Therefore, a need still exists for compositions for treating, preventing, or inhibiting ETEC infections, diseases, or disorders. In particular, a need exists for the preparation and purification of at least one CF in high purities and large amounts that are suitable for approval from governmental regulatory agencies for administration to humans.